Involvement of tyrosine kinase in citrate-stimulated aldosterone production in bovine glomerulosa cells.
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The present study was designed to assess whether citrate stimulates aldosterone production by isolated bovine adrenal glomerulosa cells in vitro. When the cells were incubated with graded concentrations of citrate up to 4.0 mM, basal aldosterone production was significantly elevated, with a gradual reduction of extracellular ionized calcium concentration. Without citrate, however, adding increasing amounts of calcium chloride to a calcium-free medium did not reproduce the citrate's effect on basal aldosterone production. Genistein, an inhibitor of tyrosine kinases, inhibited the citrate (4 mM)-induced aldosterone production in a dose-dependent manner, with 89.8% of inhibition at a concentration of 10 microM. When the cells were exposed to citrate (4 mM) for 5, 10, and 30 min, tyrosine in Mr 105,000 endogenous protein was dominantly phosphorylated. This study demonstrates for the first time that citrate stimulates aldosterone production in bovine adrenal glomerulosa cells in vitro and also suggests a crucial involvement of protein tyrosine kinase in the steroidogenic action of citrate in the cells. (+info)
Cloning and characterization of ntTMK1 gene encoding a TMK1-homologous receptor-like kinase in tobacco.
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A cDNA encoding a receptor-like kinase, designated NtTMK1, was isolated from Nicotiana tabacum. The kinase domain of NtTMK1 contained all of 12 subdomains and invariant amino acid residues found in eukaryotic protein kinases. The extracellular domain contained 11 leucine-rich repeats which have been implicated in protein-protein interactions. The amino acid sequence of NtTMK1 exhibited high homology with those of TMK1 of Arabidopsis and TMK of rice in both kinase and extracellular domains, suggesting that NtTMK1 is a TMK homologue of tobacco. The NtTMK1 transcripts were present in all major plant organs, but its level varied in different developmental stages in anthers and floral organs. NtTMK1 mRNA accumulation in leaves was stimulated by CaCl2, methyl jasmonate, wounding, fungal elicitors, chitins, and chitosan. The NtTMK1 mRNA level also increased upon infection with tobacco mosaic virus. (+info)
Interaction of the neurotoxic and nontoxic secretory phospholipases A2 with the crotoxin inhibitor from Crotalus serum.
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Crotalus durissus terrificus snakes possess a protein in their blood, named crotoxin inhibitor from Crotalus serum (CICS), which protects them against crotoxin, the main toxin of their venom. CICS neutralizes the lethal potency of crotoxin and inhibits its phospholipase A2 (PLA2) activity. The aim of the present study is to investigate the specificity of CICS towards snake venom neurotoxic PLA2s (beta-neurotoxins) and nontoxic mammalian PLA2s. This investigation shows that CICS does not affect the enzymatic activity of pancreatic and nonpancreatic PLA2s, bee venom PLA2 and Elapidae beta-neurotoxins but strongly inhibits the PLA2 activity of Viperidae beta-neurotoxins. Surface plasmon resonance and PAGE studies further demonstrated that CICS makes complexes with monomeric and multimeric Viperidae beta-neurotoxins but does not interact with nontoxic PLA2s. In the case of dimeric beta-neurotoxins from Viperidae venoms (crotoxin, Mojave toxin and CbICbII), which are made by the noncovalent association of a PLA2 with a nonenzymatic subunit, CICS does not react with the noncatalytic subunit, instead it binds tightly to the PLA2 subunit and induces the dissociation of the heterocomplex. In vitro assays performed with Torpedo synaptosomes showed a protective action of CICS against Viperidae beta-neurotoxins but not against other PLA2 neurotoxins, on primary and evoked liberation of acetylcholine. In conclusion, CICS is a specific PLA2 inhibitor of the beta-neurotoxins from the Viperidae family. (+info)
Fluid and electrolyte supplementation for exercise heat stress.
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During exercise in the heat, sweat output often exceeds water intake, resulting in a body water deficit (hypohydration) and electrolyte losses. Because daily water losses can be substantial, persons need to emphasize drinking during exercise as well as at meals. For persons consuming a normal diet, electrolyte supplementation is not warranted except perhaps during the first few days of heat exposure. Aerobic exercise is likely to be adversely affected by heat stress and hypohydration; the warmer the climate the greater the potential for performance decrements. Hypohydration increases heat storage and reduces a person's ability to tolerate heat strain. The increased heat storage is mediated by a lower sweating rate (evaporative heat loss) and reduced skin blood flow (dry heat loss) for a given core temperature. Heat-acclimated persons need to pay particular attention to fluid replacement because heat acclimation increases sweat losses, and hypohydration negates the thermoregulatory advantages conferred by acclimation. It has been suggested that hyperhydration (increased total body water) may reduce physiologic strain during exercise heat stress, but data supporting that notion are not robust. Research is recommended for 3 populations with fluid and electrolyte balance problems: older adults, cystic fibrosis patients, and persons with spinal cord injuries. (+info)
Complete protection by alpha-crystallin of lens sorbitol dehydrogenase undergoing thermal stress.
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Sorbitol dehydrogenase (l-iditol:NAD(+) 2-oxidoreductase, E.C. 1.1.1. 14) (SDH) was significantly protected from thermally induced inactivation and aggregation by bovine lens alpha-crystallin. An alpha-crystallin/SDH ratio as low as 1:2 in weight was sufficient to preserve the transparency of the enzyme solution kept for at least 2 h at 55 degrees C. Moreover, an alpha-crystallin/SDH ratio of 5:1 (w/w) was sufficient to preserve the enzyme activity fully at 55 degrees C for at least 40 min. The protection by alpha-crystallin of SDH activity was essentially unaffected by high ionic strength (i.e. 0.5 m NaCl). On the other hand, the transparency of the protein solution was lost at a high salt concentration because of the precipitation of the alpha-crystallin/SDH adduct. Magnesium and calcium ions present at millimolar concentrations antagonized the protective action exerted by alpha-crystallin against the thermally induced inactivation and aggregation of SDH. The lack of protection of alpha-crystallin against the inactivation of SDH induced at 55 degrees C by thiol blocking agents or EDTA together with the additive effect of NADH in stabilizing the enzyme in the presence of alpha-crystallin suggest that functional groups involved in catalysis are freely accessible in SDH while interacting with alpha-crystallin. Two different adducts between alpha-crystallin and SDH were isolated by gel filtration chromatography. One adduct was characterized by a high M(r) of approximately 800,000 and carried exclusively inactive SDH. A second adduct, carrying active SDH, had a size consistent with an interaction of the enzyme with monomers or low M(r) aggregates of alpha-crystallin. Even though it had a reduced efficiency with respect to alpha-crystallin, bovine serum albumin was shown to mimic the chaperone-like activity of alpha-crystallin in protecting SDH from thermal denaturation. These findings suggest that the multimeric structural organization of alpha-crystallin may not be a necessary requirement for the stabilization of the enzyme activity. (+info)
The amino-acid sequence of the abalone (Haliotis laevigata) nacre protein perlucin. Detection of a functional C-type lectin domain with galactose/mannose specificity.
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Perlucin isolated from abalone nacre consists of 155 amino acids including a glycosylated asparagine. The sequence of the first 130 amino acids shows a high similarity to the C-type carbohydrate-recognition domains of asialoglycoprotein receptors and other members of the group of C-type lectins but also a weaker similarity to related proteins without carbohydrate-binding activity. This C-type module is followed by a short C-terminal domain containing two almost identical sequence repeats with a length of 10 amino acids. Solid phase assays show a divalent metal ion-dependent binding of perlucin to (neo)glycoproteins containing D-galactose or D-mannose/D-glucose indicating that perlucin is a functional C-type lectin with broad carbohydrate-binding specificity. Our results also indicate that it may be difficult to predict carbohydrate-binding specificity and the occurrence of alternative binding configurations by amino-acid sequence comparisons and homology modeling. (+info)
Further investigation of the role of calcium in human lens protein aggregation.
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High-molecular-weight (HMW) protein from human cataractous lenses, isolated by differential centrifugation, was deaggregated in 7M urea and then reaggregated in either the presence or absence of 10 mM CaCl2. Over 90% of the material reaggregated in the presence of calcium appears to have a size greater than 50 X 10(6) daltons. By contrast, only 20% to 25% of the material reaggregated in the absence of calcium has molecular weight greater than 50 X 10(6) daltons. Disulfide formation during reaggregation is unlikely in the latter experiment, since the addition of 50 mM mercaptoethanol caused no change in results. About 60% to 70% of the low-molecular-weight (LMW) protein fraction deaggregated in 7M urea buffer can be converted to HMW species in the presence of 10 mM CaCl2, when the deaggregating agent is removed. However, only 5% to 10% of this protein is converted to HMW species if the deaggregation step is eliminated. Experiments with 45 Ca indicate that whereas calcium is necessary for the formation of the HMW aggregates, only one calcium per approximately 5 X 10(5) daltons remains bound in the reaggregated material. The data suggest that although calcium may be required to induce aggregation to HMW species, it is not required to stabilize such macromolecules. SDS-polyacrylamide gel electrophoresis of the HMW species formed upon reaggregation of the dissociated HMW species with calcium indicates the presence of all the major polypeptide subunits of the original HMW species present in the lens; however, reaggregation in the absence of calcium yields HMW species lacking in the 9600 dalton component. (+info)
Chicken coagulation factor XIIIA is produced by the theca externa and stabilizes the ovarian follicular wall.
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Development of the follicle in egg-laying species such as the chicken is regulated by systemic factors as well as by the highly orchestrated interplay of differentially expressed genes within this organ. Differential mRNA display analysis of defined phases of follicle development resulted in the characterization of coagulation factor XIIIA. It is expressed and produced by cells of the theca externa in a highly regulated manner during distinct growth phases of the follicle. Transcripts for factor XIIIA are already detectable at the beginning of follicle development and peak at the end of phase 2. Protein levels, however, still increase during phase 3, peak shortly after ovulation, and persist until the postovulatory tissue is completely resorbed. Factor XIIIA is secreted as a monomer into the extracellular matrix of the theca externa and is not associated with factor XIIIB as is the case in plasma. Our data suggest that, due to its transglutaminase activity, factor XIIIA stabilizes the follicular wall by cross-linking matrix components. Thus, coagulation factor XIIIA might play a key role in coping with the massive mechanical stress exerted by the large amount of yolk accumulating during the rapid growth phase of the oocyte. (+info)